We have cloned a mitogen-induced gene from T cells, GEM, that encodes a GTP-binding protein that is a novel member of the ras superfamily. GEM protein is transiently expressed in mid-G1 between 4 and 8 hours after activation. Ras proteins and their relatives function as regulatory binary switches in the cell, cycling between active (GTP-bound) and inactive (GDP-bound) states. Several lines of evidence suggest that GEM may play a role in signal transduction during G1 progression or associated with differentiation. In order to identify the signaling pathways in which Gem is involved, we have focused our efforts on identifying proteins which are either upstream regulators or downstream effectors of Gem. We have used the yeast two-hybrid system to identify several proteins which physically interact with Gem, and the characterization of the functional signifcance of these interactions is on-going.GEM was found to associate with BCR (breakpoint cluster region protein). BCR is a multifunctional protein which contains a kinase, a guanine dissociation stimulator for rac, and GAP (GTPase activating protein) activity for rac. We have purified recombinant GEM from baculovirus and found that soluble GEM contains undetectable intrinsic GTPase activity. Therefore we anticipate that GTP hydrolysis will be highly dependent upon a GAP. We are testing the hypothesis that BCR acts as a GAP for GEM. Alternatively, GEM binding to BCR may inhibit the activity of BCR for rac. The 14-3-3 protein interacts with GEM in a GTP-independent manner in vitro and in vivo. 14-3-3 proteins recognize phosphoserine motifs and are thought to act as scaffolding proteins that may stabalize signaling complexes or regulate protein-protein interactions. The binding site of 14-3-3 in the Gem protein has been mapped to two cooperating sites in the carboxy terminus of Gem. There is no evidence that 14-3-3 binding to Gem significantly changes the subcellular localization of Gem. We are testing the hypothesis that 14- 3-3 association modulates the protein partners to which Gem binds. An additional protein found to bind Gem in a GTP-dependent manner is ROK (rho kinase). ROK is a major effector protein of Rho which is important in modulating the function of the actin cytoskeleton. Gem inhibited ROK activity in vitro. We are assaying the effect of Gem on several ROCK dependent functions in various cellular contexts. We have found that Gem expression decreases ROK dependent stress fibers and focal adhesions in fibroblasts. In addition, Gem inhibits Rho mediated colony formation in soft agar. - GTPase, Rho kinase, BCR,